Biosynthesis of peptide messengers involves limited proteolysis of precursors which can occur extracellular (e.g. complement cascade and kallikrein-kinin system) as well as intracellularly (e.g. the stored hormones, insulin, and ACTH). The realization that angiotensin is both stored as signal in brain and also generated extracellularly from plasma precursor(s) prompted the thought that all peptide messenger families might be represented in these two types of communication systems. The studies proposed here are designed to investigate this hypothesis for peptides related to neruotensin (NT) and met 5-enkephalin (ENK), peptides which have been shown to be stored within neural or endocrine cells and released by specific stimuli. Preliminary data demonstrate the enzymatic generation of MuM amounts of biologically active and immunoreactive NT and ENK (iNT, iENK) from tissue-specific precursors in 3 different systems: (a) Blood, (b) Skin/stomach, (c) Brain/intestine. Initially, we noticed that the primary structure of NT resembled that of angiotensin I and attempted to generate iNT from blood proteins using renin. Renin was negative but pepsin, a protease which can mimic renin, elevated plasma iNT and iENK one hundred thousand-fold, producing amounts (0.5-4nmo1/ml) similar to the plasma levels of angiotensinogen and bradykininogen. Furthermore, the HPLC purified iNT and iENK exhibited biologic effects on rat blood pressure, skin vascular permeabillty and gut contractility. We also discoverd that during extraction of tissues, particularly skin and stomach, iNT and iXP (Xenopsin) were generated by endogenous enzymes. The skin-derived and stomach-derived peptides differed from the blood-derived ones and displayed different biologic effects. Finally, we investigated NT itself, known to be localized to brain and intestine. Our data suggest that its biosynthesis may also involve a pepsin-related enzyme. We aim to do the following for each system: a) Identify and localize the precursor proteins; b) Isolate, sequence and do phamacology on the generated peptides; c) Identify the endogenous processing-proteases; d) Define physiological or patho-physiological situations which lead to activation of these systems. One exciting possiblity is that these newly discovered systems represent a potential for the generation of inflammatory or analgesic substances during injury.